B6 - basics of haemostasis

Question 1

○ In the absence of injury

Answer 1

§ Intact endothelium prevents platelet adherence by production of Nitrous oxide and prostacyclin
§ Keeps blood flowing smoothly with laminar flow

Question 2

Following vascular injury

Answer 2

there is reflex vasospasm of smooth muscle (transient)
§ Expose subendothelial matrix
○ Injury exposes important proteins in subendothelium that activate proteins that activate coagulation
§ VWF
§ Tissue factor

Question 3

Circulating platelets bind to subendothelial matrix (at the site of vascular injury) through platelet surface receptor

Answer 3

(glycoproteins 1b/IX/V complex) via von-Willebrand factor (VWF)

Question 4

surface receptor on platelets that binds to sub endothelial matrix

Answer 4

□ Glycoprotein 1B/IX/V complex

® Surface glycoprotein on the platelet allowing it to bind via von Willebrand factor

Question 5

other interactions contributing to platelet adherence to sub endothelial matrix

Answer 5

GP1a/IIa, GPVI binding to collagen - less important than ib/IX/V complex

Question 6

Activated platelets

Answer 6

change shape, release the contents of their granules (which contains substances such as ADP, TXA2, fibrinogen, VWF)
□ Leads to further platelet activation at the site through ADP

Question 7

platelets are activated by

Answer 7

§ Platelets are activated by collagen, VWF, thrombin, ADP

Question 8

fibrinogen receptor

Answer 8

Express glycoprotein IIb/IIIa in an active form on the platelet surface

Question 9

Glycoprotein IIb/IIIa receptor

Answer 9

§ Express glycoprotein IIb/IIIa in an active form on the platelet surface (fibrinogen receptor)
□ Glycoprotein IIb/IIIa receptor is always on the platelet surface but only exists in an activated form when the platelet is activated
□ This activated form is the main binding process through which platelets bind fibrinogen

Question 10

platelet shape change

Answer 10

□ In resting state, they are smooth disk shape
□ Become spikey cells when activated allowing for better cross binding
□ When clot matures they because flat and spread out

Question 11

purpura

Answer 11

□ Immune thrombocytopaenia purpura
® Normal bone marrow production of platelets
® Platelets are rapidly consumed by antibody mediated mechanisms
® Surface bleeding, mucosal bleeding, bruising

Question 12

• Clotting cascade (generation of fibrin clot)

Answer 12

○ Tissue factor is exposed in the subendothelial system
○ combines with factor VIIa (circulating factor in the blood) to form a small amount of FVIIa-TF complex
○ FVIIa-TF complex activates other clotting proteins leading to a small amount of thrombin production
○ Through feedback mechanisms large scale production of thrombin takes place on platelet surface
○ Thrombin converts fibrinogen to fibrin which forms the basis of the clot
○ Fibrin mesh at site of injury

Question 13

initiation of clotting cascade

Answer 13

□ Initiation of clotting occurs when disruption of the endothelium exposes activated factor VII (VIIa) in blood to tissue factor on subendothelial cells (smooth muscle cells and fibroblasts)
□ FVIIa-TF complex activates other clotting proteins (esp. FX to FXa and FIX to FIXa)
□ Small amount of thrombin is formed

Question 14

propagation of clotting

Answer 14

□ Thrombin (Factor IIa) activates platelets which release further coagulation proteins
□ Thrombin activates coagulation proteins required for it’s own production (feedback)
□ Large scale production of thrombin takes place on platelet surface
□ Thrombin catalyses fibrinogen to fibrin - forming structure of clot
□ FXIII because FXIIIa which allows for cross linking between fibrin stands

Question 15

○ Clot stabilisation

Answer 15

§ Thrombin cleaves fibrinogen to fibrin
§ Fibrin strands provide the structural integrity to the thrombus
§ Factor XIII allows cross linking of fibrin monomers
§ Fibrinolysis is activated to localise the clot to site of injury and remove clot when healing has occurred

Question 16

• Fibrinolysis (breakdown of the fibrin clot)

Answer 16

○ The process by which a clot is removed
○ Tissue plasminogen activator (tPA) activates plasminogen and converts it to active plasmin
○ Plasmin enzymatically attacks the fibrin molecule producing fibrin degradation products (FDP’s) that are cleared from the circulation by macrophages

Question 17

summary of coagulation

Answer 17

○ Platelets bind to subendothelium collagen to initiate primary closure of the vessel wall defect
○ Tissue factor in subendothelium combines with FVII in blood to form the FVIIa-TF complex
○ FVIIa-TF complex activates other clotting proteins leading to thrombin production
○ Large scale production of thrombin takes place on the platelet surface
○ Thrombin converts fibrinogen to fibrin to form cross linked fibrin clot
○ Fibrinolysis is activated to localise the clot to the site of injury

Question 18

biological amplification system

Answer 18

• Incredibly efficient: 1 mole XIa generates ~107 moles XIIa

- Clotting factors numbered I-XIII

Question 19

• Binding of FVII to tissue factor (TF) initiates coagulation

Answer 19

§ Activates
□ FXI to XIa
□ FV to Va
□ FVIII to VIIIa
§ Massive but highly focussed burst of thrombin
□ Rapidly converts soluble fibrinogen to insoluble fibrin
□ Fibrin reinforces and stabilises the platelets ‘plug’

Question 20

contact pathway/intrinsic pathways

Answer 20

plays a lesser role

eg. deficiencies of factor 12 cause no increases tendencies of bleeding

Question 21

extrinsic/tissue pathway

Answer 21

main way of coagulation cascade activation in vivo

Question 22

if PT is normal

Answer 22

but be a problem with APTT arm

Question 23

If APTT is normal

Answer 23

must be a problem with factor 7

Question 24

citrated blood

Answer 24

• citrate decalcifies the blood
  
  • calcium is a cofactor for coagulation cascade to the blood stays liquid in the tube
  • Centrifuge blood to take out platelets and cellular components
  • Add activator and platelet substitute
  • Add calcium
  • Measure time to clot formation

Question 25

Prothrombin Time (PT)

Answer 25

• Measures extrinsic and common pathways
  ○ Sensitive to abnormalities in factors VII, X, V, II and fibrinogen
  
  • Time for clot to form after addition of tissue factor (TF), phospholipid and calcium to decalcified platelet poor plasma
  • Reference range usually ~13-15 seconds but will vary between labs based on exact technique used
  • Basis for monitoring oral vitamin K antagonists (warfarin and warfarin like compounds)
  • Relationship between PT and a factor deficiency is not linear
    ○ Prolongs exponentially at lower concentrations
    ○ For PT or APTT to be prolonged, factor should be at around 30%
    ○ Significant reduction required for prolongation
  • International Normalised Ratio
    ○ The INR is a ratio of the PT of a test compared to a normal sample. This ratio is corrected for the sensitivity of the tissue factor used in the PT assay
    ○ The INR was developed to monitor patients on oral anticoagulants (warfarin)
    ○ For other area e.g. assessing severity of coagulopathy in liver disease PT should be used

Question 26

prothrombin time is sensitive to abnormalities in

Answer 26

Sensitive to abnormalities in factors VII, X, V, II and fibrinogen

Question 27

prothrombin time measures

Answer 27

common and extrinsic pathways

Question 28

what is added to decalcified platelet poor plasma

Answer 28

• Time for clot to form after addition of tissue factor (TF), phospholipid and calcium to decalcified platelet poor plasma

Question 29

• Relationship between PT and a factor deficiency is not linear

Answer 29

○ Prolongs exponentially at lower concentrations
○ For PT or APTT to be prolonged, factor should be at around 30%
○ Significant reduction required for prolongation

Question 30

• International Normalised Ratio

Answer 30

○ The INR is a ratio of the PT of a test compared to a normal sample. This ratio is corrected for the sensitivity of the tissue factor used in the PT assay
○ The INR was developed to monitor patients on oral anticoagulants (warfarin)
○ For other area e.g. assessing severity of coagulopathy in liver disease PT should be used

Question 31

Activated Partial Thromboplastin Time

Answer 31

• Measures intrinsic and common pathways
  
  • Platelet poor plasma + phospholipid + contact activator (e.g. Kaolin) + calcium
  • Deficient factor <40% before APTT prolonged - requires significant deficiencies
  • Normal range is longer than PT
  • Reference range ~27~ 3.5s

Question 32

APTT measures

Answer 32

intrinsic and common pathways

Question 33

what is added to platelet poor plasma in APTT

Answer 33

Platelet poor plasma + phospholipid + contact activator (e.g. Kaolin) + calcium

Question 34

is APTT or PT generally longer

Answer 34

APTT

Question 35

Fibrinogen

Answer 35

• Reference range 2-4g/L
  
  • Quantitative and qualitative defects
  • Functional and immunological methods to measure
  • Functional (Clauss assay)
    ○ Clot diluted plasma with very high concentration of thrombin
    ○ Use diluted plasma to remove effects of inhibitory substances e.g. heparin and FDPs
    ○ Measures conversion of fibrinogen to fibrin
    ○ High thrombin concentration so clotting time independent of thrombin concentration for wide range of fibrinogen levels
  • Immunological methods
    ○ Measure concentration not functional activity

Question 36

• Functional (Clauss assay)

Answer 36

○ Clot diluted plasma with very high concentration of thrombin
○ Use diluted plasma to remove effects of inhibitory substances e.g. heparin and FDPs
○ Measures conversion of fibrinogen to fibrin
○ High thrombin concentration so clotting time independent of thrombin concentration for wide range of fibrinogen levels

Question 37

if both PT and APTT are prolonged

Answer 37

deficiency in factor 2, 5, 10
reduced or abnormal fibrinogen
liver disease, fit K deficiency, heparin, warfarin